Organic Chemistry

970 CHAPTER 23 Amino Acids, Peptides, and Proteins

WATER SOFTENERS:

EXAMPLES OF CATION-

EXCHANGE CHROMATOGRAPHY

Water softeners contain a column with a cation-exchange resin

that has been flushed with concentrated sodium chloride. In

Section 17.13, we saw that the presence of calcium and magne-

sium ions in water is what causes the water to be “hard.”When

water passes through the column, the resin binds magnesium

and calcium ions more tightly than it binds sodium ions. In this

way, the water softener removes magnesium and calcium ions

from water, replacing them with sodium ions. The resin must be

recharged from time to time by flushing it with concentrated

sodium chloride to replace the bound magnesium and calcium

ions with sodium ions.

SO 3 Na+ SO 3 Na+ SO 3 Na+

SO 3 Na+ SO 3 Na+

CH CH 2

CH CH 2 CH CH 2

CH 2 CH CH 2 CH CH CH 2

CH 2

CH 2 CH

CH CH 2 CH CH 2 CH CH 2 CH

CH CH 2 CH

Figure 23.3N

A section of a cation-exchange

resin. This particular resin is called

Dowex 50.®

Cations bind most strongly

to cation-exchange resins.

Anions bind most strongly

to anion-exchange resins.

Ion-Exchange Chromatography

Electrophoresis and thin-layer chromatography are analytical separations—small

amounts of amino acids are separated for analysis. Preparative separation, in which

larger amounts of amino acids are separated for use in subsequent processes, can be

achieved using ion-exchange chromatography. This technique employs a column

packed with an insoluble resin. A solution of a mixture of amino acids is loaded onto

the top of the column and eluted with a buffer. The amino acids separate because they

flow through the column at different rates, as explained below.

The resin is a chemically inert material with charged side chains. One commonly

used resin is a copolymer of styrene and divinylbenzene with negatively charged sul-

fonic acid groups on some of the benzene rings (Figure 23.3). If a mixture of lysine

and glutamate in a solution with a pH of 6 were loaded onto the column, glutamate

would travel down the column rapidly because its negatively charged side chain

would be repelled by the negatively charged sulfonic acid groups of the resin. The

positively charged side chain of lysine, on the other hand, would cause that amino

acid to be retained on the column. This kind of resin is called a cation-exchange

resinbecause it exchanges the counterions of the groups for the positively

charged species that are added to the column. In addition, the relatively nonpolar

nature of the column causes it to retain nonpolar amino acids longer than polar amino

acids. Resins with positively charged groups are called anion-exchange resins

because they impede the flow of anions by exchanging their negatively charged coun-

terions for negatively charged species that are added to the column. A common anion-

exchange resin (Dowex®1) has groups in place of the

groups in Figure 23.3.

CH 2 N+(CH 3 ) 3 Cl- SO 3 - Na+

Na+ SO 3 -

An amino acid analyzeris an instrument that automates ion-exchange chromatog-

raphy. When a solution of an amino acid mixture passes through the column of an

amino acid analyzer containing a cation-exchange resin, the amino acids move

through the column at different rates, depending on their overall charge. The solution

leaving the column is collected in fractions, which are collected often enough that a

different amino acid ends up in each fraction (Figure 23.4). If ninhydrin is added to

each of the fractions, the concentration of the amino acid in each fraction can be

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